P412: LOW EXPOSURE, EXTENDED DOSING MIMICKING CLINICAL EXPOSURES OF ORAL AZACITIDINE SYNERGIZES WITH VENETOCLAX IN PRECLINICAL AML MODELS
Authors: D. Jeyaraju, M. Alapa, A. Polonskaia, A. Risueno Perez, R. Hurren, X. Wang, M. Gronda, A. Ahsan, C. Wang, P. Subramanyam, J. Sriganesh, A. Anand, M. Bysani Reddy, K. Ghosh, C. Kyriakopoulos, N. Lailler, C. Hartl, D. Lopes de Menezes, A. Schimmer, P. Hagner, A. Gandhi, A. Thakurta
Journal: HemaSphere (2022)

Background: Acute myeloid leukemia (AML) has a high incidence of relapse despite the advancement of novel therapies. AML patients that are ineligible for intensive chemotherapy are typically administered injectable azacitidine (AZA) at a dose of 75 mg/m2 for 7 days in combination with BCL-2 inhibitor venetoclax (VEN) as induction therapy. Injectable-AZA dose was determined primarily through safety, tolerability, and bioavailability studies based on the CALGB and AZA-001 trials and not through a dose escalation study. Thus, it is not known if similar efficacy and combinability of AZA with VEN could be achieved at lower exposures and extended duration with oral-AZA dosing. The approval of oral-AZA in AML maintenance therapy, albeit at a lower cumulative exposure conferred at doses of 300 mg for an extended duration of 14 days of a 28-day dosing cycle, creates opportunities to answer these questions. Aims: To investigate the mechanism of action and combinability of oral-AZA with VEN in pre-clinical models. Methods: For in vitro cell-line based work, 1 day x 1mM AZA and 5days x 0.2mM AZA were used to mimic injectable-AZA like and oral-AZA like regimens respectively. For in vivo studies we modeled injectable-AZA and oral-AZA doses with 3mg/kg, qd x 5 days and 1 mg/kg, qd x 15 days respectively (intraperitonially in both cases, modified from Vu et al., Nat. Commun. 2020). VEN was administered orally at 100 mg/kg, qd x21 days. CRISPR screen was conducted with OCI-AML2-Cas9 cells using the Cellecta sgRNA library in the presence of AZA (121nm (IC50) x 5 days). Results: To identify synthetic lethal targets of oral-AZA, we conducted a genome-wide CRISPR screen. Among others, BCL-2 emerged as a synergistic hit. During in vitro validation of the CRISPR screen data, VEN synergized (anti-proliferative effect) with oral-AZA like exposure only when added after 5 days (0.2mM /day) of repeated AZA dosing. On the other hand, injectable-AZA like exposure synergized with VEN in 24 hours suggesting that the Oral-AZA/VEN synergy was driven more through epigenetic changes and not by stress response as has been reported for injectable-AZA. To further validate the CRISPR screen findings, we utilized in vivo AML xenograft mouse (injected with human AML cell lines) treated with an oral-AZA like or injectable-AZA like regimen with/without VEN. Mouse survival data demonstrated that both AZA regimens combined with VEN with equivalent efficacy. The persistence of leukemic stem cells (LSCs) is one of the reasons for relapse in AML. In vitro, oral-AZA like exposure resulted in decreased numbers of CD34+ CD38- LSCs. In an in vivo primary AML xenograft model using intrafemoral engraftment, oral-AZA like exposure was effective in reducing engraftment of leukemic cells from the right to the left femur (10-fold reduction). Analysis of cells at the site of injection (right femur) revealed induction of differentiation from a granulocyte-monocyte progenitor (GMP) to a megakaryocyte-erythrocyte progenitor (MEP) or common myeloid progenitor (CMP) state in response to oral-AZA. Summary/Conclusion: Collectively, our pre-clinical in vitro and in vivo data in AML models indicates that oral-AZA can combine effectively with VEN. In addition, oral-AZA can effectively eliminate LSCs and induce differentiation of immature cells. Through the CRISPR screen, our work identifies potential novel combination partners for oral-AZA.